Voltage-gated potassium channels (Kv) maintain the resting membrane potential and the shape of the action potential. The activity of these channels modulates cardiac pacemaking, the action potential duration and neurotransmitter release. In addition, these channels play an important role in oxygen-sensing, cell-volume regulation, and apoptosis. The ion-conducting pore of these channels is formed by homomeric or heteromeric assembly of Kv proteins. The cytoplasmic domain of several Kv proteins interacts with ancillary 2-subunits that belong to the aldo-keto reductase (AKR) superfamily. The Kv2 proteins display high affinity pyridine nucleotide binding and catalyze aldehyde reduction. The general physiological role of Kv2, however, remains unclear and the contribution of the AKR properties of Kv2 to the regulation of Kv channel activity has not been assessed. We propose that the binding of pyridine nucleotides to the AKR domain of the 2-subunit imparts metabolic sensitivity to Kv channels, such that an increase in the NAD(P)H/NAD(P)+ ratio increases Kv inactivation; thus coupling Kv channel activity to the metabolic state of the heart. Because the ratio of reduced and oxidized nucleotides is tightly controlled by glycolysis and mitochondrial respiration, we suggest that differential regulation of Kv1-2 currents by pyridine nucleotides is a physiological mechanism that couples myocardial excitation to changes in intermediary metabolism during hypoxia, ischemia, and apoptosis. To test this hypothesis, we will (S.Aim 1) delineate the contribution of pyridine nucleotides to the regulation of Kv currents by Kv2; (S.Aim 2) examine the metabolic dependence of Kv2-induced-inactivation of Kv currents; and (S.Aim 3) elucidate the role of Kv2 in regulating native Kv channels in the heart. To assess the significance of nucleotide binding at the aldo-keto reductase domain of Kv2 proteins, we will determine how changes in the redox state of pyridine nucleotides or the nucleotide binding affinity, induced by site-directed mutagenesis, affects the ability of Kv22 to interact with or alter Kv1.5 currents. To examine the metabolic-dependence of Kv2-inactivation of Kv currents, we will investigate how changes in metabolism or oxygen concentration affect Kv1-2 currents. For this, we will measure Kv currents from cells subjected to hypoxia or treated with metabolic inhibitors and determine whether differential pyridine nucleotide binding to Kv2 is essential for imparting metabolic and oxygen sensitivity to Kv1.5 currents. To determine how the redox properties of Kv2 affect the pathophysiological responses of Kv channels, we will examine how metabolic inhibitors or components of ischemia affect Kv currents in cardiac myocytes isolated from wild-type, Kv2-null and Kv22:Y90F mice, and whether loss of Kv2 affects tissue injury and dysfunction due to acute myocardial infarction. Successful completion of this study will provide insights in to the Kv channel function and how these channels respond to changes in myocardial oxgyen levels and metabolism. These findings could provide insights into the development of new therapeutic interventions for the treatment and management of myocardial ischemic injury.